Regulation of neural crest derived progenitor cell niche by epithelial cell derived extracellular vesicles

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Overall program goal The overall goal of this research program is to unravel the mechanisms that regulate the niche and function of oral/gingival neural crest derived stromal stem cells (NCSSCs). The NCSSCs are retained in a few specific locationssuch as bone marrow, hair follicles and oral mucosain adult tissue and their numbers generally decline with age. Using Wnt1-CRE/R26RYFP reporter mice, we have shown using tracing experiments that gingival NCSSCs are enriched in the gingival connective tissue. Interestingly, the NCSSCs are enriched at the connective tissue papilla and more specifically in close proximity to gingival oral epithelial cells (OECs). It is well known that connective tissue and epithelial cells engage in molecular crosstalk that can regulate the functions of both cell types. The crosstalk between OECs and NCSSCs is, therefore, likely to be involved in the regulation of NCSSC niche and function. There is no information available in the literature on how OECs could regulate NCSSC niche or fate. In the present proposal, we are proposing a new paradigm that OECs release extracellular vesicles that participate in the regulation of NCSSC fate. General hypothesis The general hypothesis is that signals derived from adjacent OECs regulate the fate and niche of NCSSCs in adult gingival tissue. Because the signals need to be specific and protected from off-target effects, we further hypothesize that the OECNCSSC crosstalk is regulated by extracellular vesicles (EVs). The EVs, including exosomes (EXO) and microvesicles (MVs), provide safety and receptor-mediated specificity to this regulation. Objectives and methodology The objective of the proposal is to find out how OEC-derived EXOs and MVs regulate the function and expression of differentiation-related genes and proteins in NCSSCs. Furthermore, we will investigate how these vesicles regulate the specific differentiation pathways, including osteogenic, adipogenic, chondrogenic and neuronal lineages. We will use a NCSSC cell line (named Ginestin, with over 90% nestin-positive cells) that we have isolated from adult human gingiva as our target cell line, expose it to OEC vesicles and measure the output with RT-qPCR, Western blotting, proteomics, immunostainings and other microscopic techniques. Expected outcomes and significance This research program is expected to significantly increase our understanding of how neural crest derived cells can survive in certain microenvironments such as oral mucosa. The novel aspect is the way we expect the OECs to contribute to the formation of the niche and regulation of the gene expression profile that is conductive for cell survival.